(Egr) 2 and 3 control inflammatory responses of tolerant T cells Becky Omodho

188 Pages·2016·6.17 MB·English

Early growth response gene (Egr) 2
and 3 control inflammatory
responses of tolerant T cells

A thesis submitted for the degree of Doctor of Philosophy by

Becky Omodho

April 2016
Division of Biosciences
College of Health and Life Sciences; Department of Life Science
Declaration
I hereby declare that the work completed in this thesis is my own
work unless otherwise stated, and has not been submitted for any
other degree.

Becky Omodho

i
Abstract
Abstract
This study investigated the role of tolerance induction in an inflammatory setting in regard to the
early growth response genes Egr2 and Egr3. T cells robustly respond to pathogenic antigens during
infection, but are tolerant to stimulation by self-antigens. The intrinsic mechanisms for self-tolerance
in the periphery are still not clear. Egr2 and 3 are induced in tolerant T cells in response to antigen
stimulation by NFAT-medicated tolerant signalling; however, their function in tolerant T cells is still
unknown.
The study demonstrated that Egr2 and 3, induced in tolerant T cells, are not directly involved in
defective proliferation and IL-2 production, the hallmarks of T cell tolerance. However, they are
essential for preventing inflammatory response of tolerant T cells. In the absence of Egr2 and 3,
tolerant T cells show impaired proliferation and production of IL-2, but produce high levels of IFN-γ,
a key inflammatory cytokine. This phenotype resembles CD4 T cells from autoimmune diseases such
as lupus which show poor proliferative response, but hyper-inflammation. Our study demonstrated,
for the first time, a distinctive mechanism to control inflammation from proliferative tolerance
regulated by Egr2 and 3, which may be an important mechanism for the control of autoimmune
diseases.

ii
Acknowledgements
Firstly, I would like to thank my supervisor Dr. Su-Ling Li for her invaluable guidance throughout this
project; I would also like to extend my thanks to Professor Ping Wang for sharing his extensive
knowledge. Thank you to Dr. Tizong Miao and Dr. Alistair Simmonds and Randeep Singh for sharing
their expertise whenever I needed it.
To Dr. Ane Ogbe, Dr. Punamdip Bhullar, Dr Sheba Adam Zahir, thank you for everything; for pushing
me, for encouraging me, for guiding me and most importantly for being my friends when I absolutely
needed it most, words cannot describe how lucky I am to have you in my life. To Ezgi Gokhan, thank
you for being so wonderful. To Joy Gacamiu Muthure, Kanyali Ilako, Anil Padia, Thuku wa Thuku,
Kennedy Kairu Mbugua and Sabrina Sondo, I would like to express to you my utmost gratitude.
Thank you for holding my hand throughout all this. Thank you for telling me what I needed to hear
and thank you for all your love, for reminding me that friendship knows no distance. I could not have
done this without your support.
To my irreplaceable family: Damaris, Arnold, Stacey and Sussie, thank you for your absolute and
unflinching support. To the Weinerts, I cannot describe to you how thankful I am for giving me a
home away from home, for your support and for so much love. To Prisca, you have been more than
a friend to me; to say I appreciate it would be a complete understatement. To Rowena, thank you
for your guidance and love and for keeping me in your thoughts. To Joseph, thank you for easing the
tough days. To my aunts Rose and Rose, thank you for your ever watchful eyes. I love you all dearly.
To my parents Rose and Wellington, thank you for absolutely everything; for giving me this
opportunity, for showing me the value of hard work, for instilling values that I live my life by and for
being strong for me when I couldn’t be. I could not have done this without you. To my mother, thank
you for inspiring me each day. Thank you for helping me realise the importance of knowing myself,
in the words of Chinua Achebe ‘Nobody can teach me who I am, you can describe parts of me but
who I am and what I need is something I have to find out for myself’.
To Chandi Mosomi, I do not know how to truly express my gratitude to you; you were by my side
through every step of the way. Thank you for your support and love and for always believing in me. I
am truly blessed to have you in my life. I love you dearly.
I thank God for guiding me throughout this process and for guiding my family and friends.
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Dedication
This is for my parents Rose and Wellington Omodho; thank you for everything you have done
for me. Thank you for teaching me that respect is earned, honesty is appreciated, trust is
gained, and happiness is a journey.
I am forever grateful for the both of you.
I love you.
Table of contents

Table of contents
Declaration i
Abstract ii
Acknowledgements ...................................................................................... iii
Dedication iv
Table of contents ........................................................................................... v
Table of figures................................................................................................................................. viii
Table of tables .................................................................................................................................. xiii
Abbreviations ....................................................................................................................................xiv
Chapter 1. Introduction ............................................................................... 1
1.1 Governance of the immune system ........................................................................................... 1
1.2 Innate immunity ......................................................................................................................... 1
1.2.1 Cells of innate immunity..................................................................................................... 1
1.2.2 Cytokines ............................................................................................................................ 1
1.2.3 Adaptive immunity ............................................................................................................. 3
1.3 Central tolerance ........................................................................................................................ 5
1.3.1 T cell development ............................................................................................................. 5
1.4 Peripheral T cell tolerance ....................................................................................................... 18
1.4.1 T cell Activation ................................................................................................................ 18
1.4.2 T cell differentiation ......................................................................................................... 25
1.4.3 Extrinsic mechanisms ....................................................................................................... 35
1.5 Autoimmunity .......................................................................................................................... 37
1.5.1 Mechanisms of tolerance breakdown .............................................................................. 37
1.5.2 Systemic and organ specific autoimmune disease ........................................................... 40
1.5.3 Autoimmune diseases with inflammation ....................................................................... 41
1.6 Experimental settings for tolerance induction ........................................................................ 41
1.6.1 Induction of CD4 T cell clonal anergy in vitro ................................................................... 41
1.6.2 In-vivo tolerance induction .............................................................................................. 42
1.7 Early growth response (Egr) genes........................................................................................... 45
1.7.1 Egr family .......................................................................................................................... 45
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1.7.2 Egr function ...................................................................................................................... 46
1.7.3 Egr2 knockout model ........................................................................................................ 46
1.7.4 Egr2 and Egr3 knockout model ........................................................................................ 47
1.7.5 Egr2 in autoimmune disease ............................................................................................ 48
1.7.6 Egr2 and 3 in tolerance ..................................................................................................... 49
1.8 Aim of study ............................................................................................................................. 50
Chapter 2. General Methods and Materials ............................................... 51
2.1 Generation of Egr2-/-Egr3-/- mice ........................................................................................... 51
2.1.1 Animal housing and care .................................................................................................. 51
2.1.2 CD2 specific Egr2 knockout mice (CD2-Egr2-/-) ............................................................... 51
CD2 specific Egr2-/-Egr3-/- mice. ..................................................................................................... 52
2.2 Genotyping ............................................................................................................................... 52
2.3 Organ collection and preparation ............................................................................................ 54
2.4 Haematoxylin and Eosin (H&E) staining ................................................................................... 55
2.5 Immunohistochemistry of frozen sections .............................................................................. 55
2.6 Isolation of CD4 T cells ............................................................................................................. 55
2.7 Tolerance induction ................................................................................................................. 56
2.7.1 In-vitro CD4+ T cell anergy induction ............................................................................... 56
2.7.2 In-vivo tolerance induction .............................................................................................. 57
2.8 Cell viability .............................................................................................................................. 58
2.9 Enzyme Linked Immunosorbent Assay (ELISA) ........................................................................ 58
2.9.1 Interleukin 2 (IL-2) ELISA .................................................................................................. 58
2.9.2 Interferon gamma (IFN-γ) ELISA ....................................................................................... 59
2.10 Flow cytometry ........................................................................................................................ 59
2.10.1 Flow cytometry antibodies ............................................................................................... 60
2.11 Proliferation measurement ...................................................................................................... 60
2.12 Electronic mobility shift assay (EMSA) ..................................................................................... 61
2.12.1 EMSA protein extraction .................................................................................................. 61
2.12.2 Bradford assay-protein quantification ............................................................................. 61
2.12.3 EMSA Binding reactions ................................................................................................... 62
2.13 RNA extraction ......................................................................................................................... 62
2.14 cDNA production ...................................................................................................................... 63
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2.15 Primer design ........................................................................................................................... 63
2.16 Real Time PCR .......................................................................................................................... 64
2.17 RT-PCR data analysis ................................................................................................................ 65
2.18 Statistical analysis .................................................................................................................... 66
Chapter 3. In-vitro tolerogenic induction in Egr2 and Egr3 knockout CD4 T
cells .......................................................................................... 67
3.1 Introduction ............................................................................................................................. 67
3.2 Methods ................................................................................................................................... 68
3.2.1 Organ collection, preparation and staining ...................................................................... 68
3.2.2 CD4 T cell isolation ........................................................................................................... 68
3.2.3 Cell stimulation ................................................................................................................. 69
3.2.4 EMSA-electronic mobility shift assay ............................................................................... 70
3.3 Results ...................................................................................................................................... 70
3.3.2 The effect of anergy induction in Egr2-/-Egr3-/- mice ..................................................... 82
3.3.3 The impact of TCR signalling on Egr2 expression in the tolerance setting ...................... 87
3.3.4 Gene expression during in vitro tolerance induction ....................................................... 99
3.4 Discussion ............................................................................................................................... 107
Chapter 4. In-vivo tolerogenic induction in Egr2 and Egr3 knockout CD4 T
cells ........................................................................................ 111
4.1 Introduction ........................................................................................................................... 111
4.2 Methods ................................................................................................................................. 112
4.2.1 In-vivo mouse treatment ................................................................................................ 112
4.2.2 Ex-vivo cell culture .......................................................................................................... 112
4.3 Results .................................................................................................................................... 113
4.3.2 Phenotypic identification of in vivo tolerised T cells ...................................................... 120
4.3.3 Inflammatory cytokine production within in vivo tolerant CD4 T cells .......................... 126
4.3.4 T-bet induction in anergic T cells .................................................................................... 129
4.4 Discussion ............................................................................................................................... 133
Chapter 5. General Discussion ................................................................. 136
5.1 Tolerance induction in the absence of Egr2 and Egr3 ........................................................... 136
5.2 Egr2, Egr3 and inflammation in the tolerant setting ............................................................. 139
5.3 Varying Egr2 expression in autoimmune disease .................................................................. 142
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Chapter 6. Conclusions, limitations and future work ............................... 144
References 149
APPENDIX 170

Table of figures
Figure 1: Innate and Adaptive Immunity ................................................................................................ 4
Figure 2: T cell development in the thymus ........................................................................................... 6
Figure 3: αβ T cell receptor structure ..................................................................................................... 7
Figure 4: Major histocompatibility complex (MHC) Structure ................................................................ 9
Figure 5: T lymphocyte fate .................................................................................................................. 10
Figure 6: Affinity thresholds in T cell selection ..................................................................................... 11
Figure 7 : Positive selection in T lymphocytes ...................................................................................... 12
Figure 8: Negative selection .................................................................................................................. 15
Figure 9: Bcl-2 apoptosis pathway ........................................................................................................ 17
Figure 11: Signals required for T lymphocyte activation ...................................................................... 19
Figure 12: CD28 in T cell interactions ................................................................................................... 22
Figure 13: T cell activation transcription network ................................................................................ 24
Figure 14: T helper cell subsets ............................................................................................................. 27
Figure 15: Mechanisms of T cell plasticity ............................................................................................ 28
Figure 16: Mechanism of CD4+ T cell anergy ........................................................................................ 33
Figure 17: Production of T regulatory T cells ........................................................................................ 36
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Figure 18: Tolerance breakdown mechanisms ..................................................................................... 40
Figure 19: Comparison of TCR binding and superantigen binding ....................................................... 43
Figure 20: The role of Egr2 and Egr3 in T cells ...................................................................................... 48
Figure 21 - Cre-Loxp system .................................................................................................................. 51
Figure 22: Phases of PCR amplification curve ....................................................................................... 66
Figure 23: In-vitro tolerance induction ................................................................................................. 69
Figure 24: PCR verification of CD2-Egr2-/-Egr3-/- Mice ........................................................................ 71
Figure 25: Confirmation of Egr2 Protein WT and expression in Egr2-/-Egr3-/- mice ........................... 72
Figure 26: Deficiency of Egr2 and Egr3 impairs proliferative responses of CD4 T cells ........................ 74
Figure 27: Deficiency of Egr2 and Egr3 impaired IL-2 production of CD4 T cells .................................. 75
Figure 28: Visualisation of impaired proliferation levels in naïve CD4 T cells from wild type and CD2-
Egr2-/-Egr3-/- mice in response to anergic stimulation .......................................................................... 76
Figure 29: Impaired proliferative response in WT and Egr2-/-Egr3-/- CD4 T cells in response to
anergic stimulation ............................................................................................................................... 77
Figure 30: Impaired IL-2 production in WT and Egr2-/-Egr3-/- CD4 T cells in response to anergic
stimulation ............................................................................................................................................ 78
Figure 31: Negligible cell death within anergic WT and Egr2-/-Egr3-/- CD4 T cells .............................. 79
Figure 32: Cyclosporine A rescues IL-2 production in WT and Egr2-/-Egr3-/- anergic CD4 T cells ....... 80
Figure 33: Cyclosporine A recovers proliferative responses of WT and Egr2-/-Egr3-/- anergic CD4 T
cells ....................................................................................................................................................... 81
Figure 34: Deficiency of Egr2 and Egr3 results in increased IFN-γ production in CD4 T cells ............... 83
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